Fig. 4.

Reconstitution of VPO1 activity. A. Effect of NaBu and hematin on cellular peroxidase activity. Empty vector (1) or expression vector encoding VPO1 (2) was transfected into HEK 293H cells as described in Materials and Methods. The cells were cultured in 5 mM of NaBu and 1 µg/ml of hematin at 37%, 5% CO₂ for 24 hrs before harvest. The TMB oxidation was carried out using lyzed cells. Error bars show the range of three independent experiments. Inset shows a Western blot indicating VPO1 expression in one of the experiment. B. VPO1-stably expressing cells were incubated with 5 mM NaBu and 1 µg/ ml hematin for indicated time. Western blotting was carried out to determine the VPO1 expression levels using anti-VPO1 antibody while the same membrane was blotted with anti-tubulin antibody as a loading control. C. Optimization of NaBu concentration. Cells were grown to 80% confluence, and 1 µg/ml hematin plus the indicated concentration of NaBu were added to the media and cells were cultured for an additional 24 hrs. TMB oxidation was carried out as described in A. GJ-3 and GJ-4 are two cell colonies that stably-expressing VPO1 and are derived from HK293H cells as described in Materials and Methods. D. Peroxidase activity in living cells. Luminol-based chemiluminescence was measured in living cells as described in Materials and Methods at the indicated concentrations of exogenously added H₂O₂. Data are representative of three independent experiments. RLU refers to relative light units. E. Inhibition of VPO1-dependent peroxidation of TMB by ABH. HEK293H and GJ-4 cells were incubated with 5 mM NaBu and 1 µg/ ml hematin for 24 hrs before harvesting. TMB oxidation was measured as described in A except that the indicated concentration of ABH was added to the incubation.